Transient receptor potential channel 1/4 reduces subarachnoid hemorrhage-induced early brain injury in rats via calcineurin-mediated NMDAR and NFAT dephosphorylation.

Transient receptor potential channel 1/4 (TRPC1/4) are considered to be related to subarachnoid hemorrhage (SAH)-induced cerebral vasospasm. In this study, a SAH rat model was employed to study the roles of TRPC1/4 in the early brain injury (EBI) after SAH. Primary cultured hippocampal neurons were exposed to oxyhemoglobin to mimic SAH in vitro. The protein levels of TRPC1/4 increased and peaked at 5 days after SAH in rats. Inhibition of TRPC1/4 by SKF96365 aggravated SAH-induced EBI, such as cortical cell death (by TUNEL staining) and degenerating (by FJB staining). In addition, TRPC1/4 overexpression could increase calcineurin activity, while increased calcineurin activity could promote the dephosphorylation of N-methyl-D-aspartate receptor (NMDAR). Calcineurin antagonist FK506 could weaken the neuroprotection and the dephosphorylation of NMDAR induced by TRPC1/4 overexpression. Contrarily, calcineurin agonist chlorogenic acid inhibited SAH-induced EBI, even when siRNA intervention of TRPC1/4 was performed. Moreover, calcineurin also could lead to the nuclear transfer of nuclear factor of activated T cells (NFAT), which is a transcription factor promoting the expressions of TRPC1/4. TRPC1/4 could inhibit SAH-induced EBI by supressing the phosphorylation of NMDAR via calcineurin. TRPC1/4-induced calcineurin activation also could promote the nuclear transfer of NFAT, suggesting a positive feedback regulation of TRPC1/4 expressions.

Melatonin attenuated early brain injury induced by subarachnoid hemorrhage via regulating NLRP3 inflammasome and apoptosis signaling.

Subarachnoid hemorrhage (SAH) is a devastating condition with high morbidity and mortality rates due to the lack of effective therapy. Nucleotide-binding oligomerization domain-like receptor family pyrin domain-containing 3 (NLRP3) inflammasome activation associated with the upregulation of apoptotic signaling pathway has been implicated in various inflammatory diseases including hemorrhagic insults. Melatonin is reported to possess substantial anti-inflammatory properties, which is beneficial for early brain injury (EBI) after SAH. However, the molecular mechanisms have not been clearly identified. This study was designed to investigate the protective effects of melatonin against EBI induced by SAH and to elucidate the potential mechanisms. The adult mice were subjected to SAH. Melatonin or vehicle was injected intraperitoneally 2 hr after SAH. Melatonin was neuroprotective, as shown by increased survival rate, as well as elevated neurological score, greater survival of neurons, preserved brain glutathione levels, and reduced brain edema, malondialdehyde concentrations, apoptotic ratio, and blood-brain barrier (BBB) disruption. Melatonin also attenuated the expressions of NLRP3, apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC), cleaved caspase-1, interleukin-1ß (IL-1ß), and interleukin-6 (IL-6); these changes were also associated with an increase in the anti-apoptotic factor (Bcl2) and reduction in the pro-apoptotic factor (Bim). In summary, our results demonstrate that melatonin treatment attenuates the EBI following SAH by inhibiting NLRP3 inflammasome-associated apoptosis.

Sulfonylurea Receptor 1 in Humans with Post-Traumatic Brain Contusions.

Post-traumatic brain contusions (PTBCs) are traditionally considered primary injuries and can increase in size, generate perilesional edema, cause mass effect, induce neurological deterioration, and cause death. Most patients experience a progressive increase in pericontusional edema, and nearly half, an increase in the hemorrhagic component itself. The underlying molecular pathophysiology of contusion-induced brain edema and hemorrhagic progression remains poorly understood. The aim of this study was to investigate sulfonylurea 1/transient receptor potential melastatin 4 (SUR1-TRPM4) ion channel SUR1 expression in various cell types (neurons, astrocytes, endothelial cells, microglia, macrophages, and neutrophils) of human brain contusions and whether SUR1 up-regulation was related to time postinjury. Double immunolabeling of SUR1 and cell-type- specific proteins was performed in 26 specimens from traumatic brain injury patients whose lesions were surgically evacuated. Three samples from limited brain resections performed for accessing extra-axial skull-base tumors or intraventricular lesions were controls. We found SUR1 was significantly overexpresed in all cell types and was especially prominent in neurons and endothelial cells (ECs). The temporal pattern depended on cell type: 1) In neurons, SUR1 increased within 48 h of injury and stabilized thereafter; 2) in ECs, there was no trend; 3) in glial cells and microglia/macrophages, a moderate increase was observed over time; and 4) in neutrophils, it decreased with time. Our results suggest that up-regulation of SUR1 in humans point to this channel as one of the important molecular players in the pathophysiology of PTBCs. Our findings reveal opportunities to act therapeutically on the mechanisms of growth of traumatic contusions and therefore reduce the number of patients with neurological deterioration and poor neurological outcomes.

Pharmacological induction of the 70-kDa heat shock protein protects against brain injury.

The 70-kDa heat shock protein (HSP70) is known to protect the brain from injury through multiple mechanisms. We investigated the effect of pharmacological HSP70 induction in experimental traumatic brain injury (TBI). 3-month-old male C57/B6 mice were given 17-N-allylamino-17-demethoxygeldanamycin (17-AAG) intraperitoneally (IP, 2 mg/kg) or intracerebroventricularly (ICV, 1 µg/kg) to determine whether HSP70 could be induced in the brain. Mice were subjected to TBI via cortical controlled impact, and were treated with 17-AAG (or vehicle) IP according to one of two treatment regimens: (1) 2 mg/kg at the time of injury, (2) a total of three doses (4 mg/kg) at 2 and 1d prior to TBI and again at the time of injury. Brains were assessed for HSP70 induction, hemorrhage volume at 3 d, and lesion size at 14 d post-injury. Immunohistochemistry showed that both IP and ICV administration of 17-AAG increased HSP70 expression primarily in microglia and in a few neurons by 24 h but not in astrocytes. 17-AAG induced HSP70 in injured brain tissue as early as 6 h, peaking at 48 h and largely subsiding by 72 h after IP injection. Both treatment groups showed decreased hemorrhage volume relative to untreated mice as well as improved neurobehavioral outcomes. These observations indicate that pharmacologic HSP70 induction may prove to be a promising treatment for TBI.

Traumatic brain injury and its effect on coagulopathy.

Polytraumatic injury results in tissue factor (TF) release from damaged cells. The acute coagulopathy of trauma (ACT) occurs early and results from significant tissue injury and tissue hypoperfusion. ACT is augmented by therapies resulting in acidemia, hypothermia, and hemodilution contributing to trauma-induced coagulopathy. Coagulopathy associated with traumatic brain injury (TBI) results from the interplay of numerous variables. Because of the high concentration of TF in brain tissue, TBI has been believed to be associated with a greater degree of coagulopathy compared with injury in other body systems. TBI has also recently been shown to cause platelet dysfunction. Platelet receptor inhibition prevents cellular initiation and amplification of the clotting cascade, limiting thrombin incorporation, and stabilization of clot to stop hemorrhage. Therefore, head injury in the presence of polytrauma does appear to augment ACT and warrants close monitoring and appropriate intervention.

Mouse brain PSA-NCAM levels are altered by graded-controlled cortical impact injury.

Traumatic brain injury (TBI) is a worldwide endemic that results in unacceptably high morbidity and mortality. Secondary injury processes following primary injury are composed of intricate interactions between assorted molecules that ultimately dictate the degree of longer-term neurological deficits. One comparatively unexplored molecule that may contribute to exacerbation of injury or enhancement of recovery is the posttranslationally modified polysialic acid form of neural cell adhesion molecule, PSA-NCAM. This molecule is a critical modulator of central nervous system plasticity and reorganization after injury. In this study, we used controlled cortical impact (CCI) to produce moderate or severe TBI in the mouse. Immunoblotting and immunohistochemical analysis were used to track the early (2, 24, and 48 hour) and late (1 and 3 week) time course and location of changes in the levels of PSA-NCAM after TBI. Variable and heterogeneous short- and long-term increases or decreases in expression were found. In general, alterations in PSA-NCAM levels were seen in the cerebral cortex immediately after injury, and these reductions persisted in brain regions distal to the primary injury site, especially after severe injury. This information provides a starting point to dissect the role of PSA-NCAM in TBI-related pathology and recovery.

Key role of sulfonylurea receptor 1 in progressive secondary hemorrhage after brain contusion.

An important but poorly understood feature of traumatic brain injury (TBI) is the clinically serious problem of spatiotemporal progression ("blossoming") of a hemorrhagic contusion, a phenomenon we term progressive secondary hemorrhage (PSH). Molecular mechanisms of PSH are unknown and efforts to reduce it by promoting coagulation have met with equivocal results. We hypothesized that PSH might be due to upregulation and activation of sulfonylurea receptor 1 (SUR1)-regulated NC(Ca-ATP) channels in capillary endothelial cells, predisposing to oncotic death of endothelial cells and catastrophic failure of capillary integrity. Anesthetized adult male rats underwent left parietal craniectomy for induction of a focal cortical contusion. The regulatory subunit of the channel, SUR1, was prominently upregulated in capillaries of penumbral tissues surrounding the contusion. In untreated rats, PSH was characterized by progressive enlargement of the contusion deep into the site of cortical impact, including corpus callosum, hippocampus, and thalamus, by progressive accumulation of extravasated blood, with a doubling of the volume during the first 12 h after injury, and by capillary fragmentation in penumbral tissues. Block of SUR1 using low-dose (non-hypoglycemogenic) glibenclamide largely eliminated PSH and capillary fragmentation, and was associated with a significant reduction in the size of the necrotic lesion and in preservation of neurobehavioral function. Antisense oligodeoxynucleotide against SUR1, administered after injury, reduced both SUR1 expression and PSH, consistent with a requirement for transcriptional upregulation of SUR1. Our findings provide novel insights into molecular mechanisms responsible for PSH associated with hemorrhagic contusions, and point to SUR1 as a potential therapeutic target in TBI.

Late proton magnetic resonance spectroscopy following traumatic brain injury during early childhood: relationship with neurobehavioral outcomes.

We sought to extend previous research that demonstrates reduced neurometabolite concentrations during the chronic phase of pediatric traumatic brain injury (TBI) in children injured during early childhood. We hypothesized that young children with TBI in the chronic phase post-injury would have lower N-acetyl aspartate (NAA) metabolite concentrations in gray and white matter in comparison to controls. We also hypothesized that metabolite levels would be correlated with acute TBI severity and neurobehavioral skills. Ten children with a history of TBI between the ages of 3 and 6 years were compared to an age, gender, and race-matched group of 10 children with a history of an orthopedic injury (OI). Children completed neurobehavioral testing at 12 months post-injury. Proton magnetic resonance (MR) spectroscopy was completed at least 12 months post-injury when the children were 6-9 years old. Groups were compared on metabolite concentrations in the medial frontal gray matter and left frontal white matter. Metabolite levels were correlated with Glasgow Coma Scale (GCS) scores and neurobehavioral functioning. There was a trend for lower NAA concentrations in the medial frontal gray matter for the TBI group. Late NAA and Cr levels in the medial frontal gray matter and NAA levels in the left frontal white matter were strongly positively correlated with initial GCS score. Metabolite levels were correlated with some neurobehavioral measures differentially for children with TBI or OI. Some neurometabolite levels differed between the TBI and OI groups more than 1 year post-injury and were related to injury severity, as well as some neurobehavioral outcomes following TBI during early childhood.

Time course of post-traumatic mitochondrial oxidative damage and dysfunction in a mouse model of focal traumatic brain injury: implications for neuroprotective therapy.

In the present study, we investigate the hypothesis that mitochondrial oxidative damage and dysfunction precede the onset of neuronal loss after controlled cortical impact traumatic brain injury (TBI) in mice. Accordingly, we evaluated the time course of post-traumatic mitochondrial dysfunction in the injured cortex and hippocampus at 30 mins, 1, 3, 6, 12, 24, 48, and 72 h after severe TBI. A significant decrease in the coupling of the electron transport system with oxidative phosphorylation was observed as early as 30 mins after injury, followed by a recovery to baseline at 1 h after injury. A statistically significant (P<0.0001) decline in the respiratory control ratio was noted at 3 h, which persisted at all subsequent time-points up to 72 h after injury in both cortical and hippocampal mitochondria. Structural damage seen in purified cortical mitochondria included severely swollen mitochondria, a disruption of the cristae and rupture of outer membranes, indicative of mitochondrial permeability transition. Consistent with this finding, cortical mitochondrial calcium-buffering capacity was severely compromised by 3 h after injury, and accompanied by significant increases in mitochondrial protein oxidation and lipid peroxidation. A possible causative role for reactive nitrogen species was suggested by the rapid increase in cortical mitochondrial 3-nitrotyrosine levels shown as early as 30 mins after injury. These findings indicate that post-traumatic oxidative lipid and protein damage, mediated in part by peroxynitrite, occurs in mitochondria with concomitant ultrastructural damage and impairment of mitochondrial bioenergetics. The data also indicate that compounds which specifically scavenge peroxynitrite (ONOO(-)) or ONOO(-)-derived radicals (e.g. ONOO(-)+H(+) --> ONOOH --> (*)NO(2)+(*)OH) may be particularly effective for the treatment of TBI, although the therapeutic window for this neuroprotective approach might only be 3 h.

Evaluation of reference tissue models for the analysis of [11C](R)-PK11195 studies.

[(11)C](R)-PK11195 is a marker of activated microglia, which can be used to measure inflammation in neurologic disorders. The purpose of the present study was to define the optimal reference tissue model based on a comparison with a validated plasma input model and using clinical studies and Monte Carlo simulations. Accuracy and reproducibility of reference tissue models were evaluated using Monte Carlo simulations. The effects of noise and variation in specific binding, nonspecific binding and blood volume were evaluated. Dynamic positron emission tomography scans were performed on 13 subjects, and radioactivity in arterial blood was monitored online. In addition, blood samples were taken to generate a metabolite corrected plasma input function. Both a (validated) two-tissue reversible compartment model with K(1)/k(2) fixed to whole cortex and various reference tissue models were fitted to the data. Finally, a simplified reference tissue model (SRTM) corrected for nonspecific binding using plasma input data (SRTM(pl_corr)) was investigated. Correlations between reference tissue models (including SRTM(pl_corr)) and the plasma input model were calculated. Monte Carlo simulations indicated that low-specific binding results in decreased accuracy and reproducibility. In this respect, the SRTM and SRTM(pl_corr) performed relatively well. Varying blood volume had no effect on performance. In the clinical evaluation, SRTM(pl_corr) and SRTM had the highest correlations with the plasma input model (R(2)=0.82 and 0.78, respectively). SRTM(pl_corr) is optimal when an arterial plasma input curve is available. Simplified reference tissue model is the best alternative when no plasma input is available.

Susceptibility weighted imaging: neuropsychologic outcome and pediatric head injury.

Traumatic brain injury is among the most frequent pediatric neurologic disorders in the United States, affecting multiple aspects of neuropsychologic functioning. This study assessed the efficacy of susceptibility weighted imaging as a predictor of long-term neuropsychologic functioning after pediatric brain injury compared with magnetic resonance spectroscopic imaging. Susceptibility weighted imaging is a relatively new method that is considered superior to traditional magnetic resonance imaging sequences for detecting hemorrhagic diffuse axonal injury. In this study, imaging and spectroscopy were acquired 6 +/- 4 days after injury. Measures of neuropsychologic functioning were administered to 18 children and adolescents 1-4 years post injury. Negative correlations between lesion number and volume with neuropsychologic functioning were demonstrated. Lesion volume explained over 32% of the variance in cognitive performance, explaining at least an additional 20% beyond injury severity and age at injury alone and 19% beyond magnetic resonance spectroscopic metabolite variables. Exploratory analyses resulted in notable trends, with lesions in deeper brain regions more strongly associated with poorer neuropsychologic performance. Improved detection of the extent of diffuse axonal injury following a brain injury will allow for a better understanding of its association with long-term outcome, which in turn can improve prognostic efficacy for effective treatment planning.